rwsés' - ' . . .09”.~. . . . ‘09”- on... o - u a to. o o'c’ ch'.‘ .°.‘/ '.'.'.'-'-’ . ‘ ' - ' ..‘_.‘ .‘? .' OI 3‘. 0-0~.. "1' "1-1? “ff."‘h‘ - .‘ ..'. ~“- STUDY ON “FISH Mums or LAKE MANITGU, MICHIGAN 9 .. f " WITH SPECIAL REFERENCE- TO INFESTATION op. - ' SMALLMOUTH ”BASS, BY THE; BASS TAPEWORM, PROTEIOCEW. AMBLOPLITIS (LEIbY‘). f Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY Pram Shankar Prasad 1963 IIUL 1.1 8 2832 w 3 02 ‘II F' )\ Lh" Us ABSTRACT STUDY ON FISH PARASITE OF LAKE MANITOU, MICHIGAN WITH SPECIAL REFERENCE TO INFESTATION OF SMALLMOUTH BASS BY THE BASS TAPEWORM, PROTEOCEPHALUS AMBLOPLITIS (LEIDY) by Prem Shankar Prasad This is a report of an investigation of the degree of infestation of smallmouth bass of Lake Manitou, Michigan, by the bass tapeworm, Proteocephalus amblgplitis, and the extent of host tissue damage. A sample of 42 fishes was examined in this study Which was represented by 36 small- mouth bass, five yellow perch, and one green sunfish. Al- together, nine different species of helminth parasites from the three phyla were recovered. The larval stage of the bass tapeworm (plerocercoids) were present in all the 42 fishes examined and were found to be most damaging. The extent of damage is greater in the females than in the males of the same age group. A study on larval lengths revealed that gonads, especially the ovaries, are better suited for the growth of these larvae. As the fish advance in age the larvae in the gonads also increase in length. The rate of growth of larvae is approximately three times greater in Prem Shankar Prasad the females than in the males. Parasites recovered from all three species of fishes examined are listed separately. Life cycles of parasites are summarized in a different table with names of their first and second accessory hosts where they are known. Further work on the direct line of control is needed because of the damaging effects of this tapeworm. STUDY ON FISH PARASITE OF LAKE MANITOU, MICHIGAN WITH SPECIAL REFERENCE TO INFESTATION OF SMALLMOUTH BASS BY THE BASS TAPEWORM, PROTEOCEPHALUS AMBLOPLITIS (LEIDY) BY Prem Shankar Prasad A THESIS Submitted to Michigan State University in partial fulfillments of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1963 ACKNOWLEDGMENTS The author wishes to express his sincere and deep appreciation to Dr. P. I. Tack, Professor and Chairman, Department of Fisheries and Wildlife, for his expert guidance, encouragement during the entire course of this investigation, and for his invaluable assistance in the collection of data and the preparation of the manuscript. He is indebted to Dr. W. D. Lindquist, Professor, Depart- ment of Microbiology and Public Health, for his valuable suggestions. Gratitude is expressed to Dr. David T. Clark of Microbiobiology and Public Health Department for his careful reading and constructive criticism of the manuscript. Special thanks are due to members of the Manitou Island Association, North Manitou Island, Michigan, for supplying fish sample. Without this support, undertaking of this study would not have been possible. ii TABLE OF INTRODUCTION . . . . . . . . REVIEW OF THE LITERATURE . . MATERIALS AND METHODS . . . Obtaining fish sample . Handling of fish . . . . Handling of parasites . Determination of the age Identification of helminths Biometrics used . . . . RESULTS AND DISCUSSIONS . . CONTENTS of fish Proteocephalus ambloplitis . Comparison of the plerocercoid larvae in young and adult smallmouth bass glinostomum marqinatum . Posthodiplostomum (= Neascus Posthodiplostomum minimum Leptorhynchoides thecatus Pomphorynchus bulbocolli Dacnitoides cotylophora SUMMARY AND CONCLUSIONS . . LITERATURE CITED . . . . . . iii 12 12 12 14 15 16 17 18 24 4O 51 58 59 69 73 76 77 82 TABLE LIST OF TABLES Parasites Recovered from Smallmouth Bass, Micropterus dolomieui, Taken from Lake Manitou, Michigan . . . . . . . . . . . . Parasites Recovered from Yellow Perch, Perca flavescens (Mitchill), Taken from Lake Manitou, Michigan . . . . . . . . . . Parasites Recovered from Green Sunfish, Lepomis cynellus Rafinesque, Taken from Lake Manitou, Midhigan . . . . . . . . . . Descriptive Summary of Helminth Parasites with Relation to the Fish Host Taken from Lake Manitou, Michigan . . . . . . . . . . Average Length of Plerocercoid Larvae of Eroteocephalus ambloplitis in the Visceral Organs of Smallmouth Bass of Various Age Groups . . . . . . . . . . . . . . . . . . Comparison of the Plerocercoid Larvae of groteocephalus ambloplitis in the Visceral Organs of the Smallmouth Bass Irrespective of Their Age Group . . . . . . . . . . . . Correlation and Regression Coefficients Between the Lengths of Plerocercoid Larvae of the Bass Tapeworm and the Annual Incre- ment in the Age of the Smallmouth Bass . . Life Cycles of Helminth Parasites Obtained from the Fishes of Lake Manitou, Michigan iv Page 19 21 23 35 41 42 44 55 FIGURE 10. 11. LIST OF FIGURES Page Unarmed Scolex of Proteocephalus ambloplitis (adult) . . . . . . . . . . . . 26 Mature Proglottid of Bass Tapeworm, Proteo- cephalus ambloplitis, Showing the Position of Uterus, Testes, and Vitelline Glands . . 28 Larvae of Proteocephalus ambloplitis (Plerocercoid with Characteristic Ves- tigial Fifth Sucker Embedded Distally in the Evaginated Scolex . . . . . . . . . 30 Larval Stage of Bass Tapeworm, Proteocepha— lus ambloplitis, (Plerocercoid) with Everted Scolex . . . . . . . . . . . . . . 33 Section of Liver of Smallmouth Bass Showing Extensive Degeneration of the Liver Tissue 38 The Relationship Between the Age of the Fish and the Length of Plerocercoid Larvae of Proteocephalus ambloplitis Growing in the Ovaries of Smallmouth Bass . . . . . . 46 The Relationship Between the Age of Fish and the Length of Plerocercoid Larvae of Proteocephalus ambloplitis Growing in the Testes of Smallmouth Bass . . . . . . . . . 48 Metacercaria of Clinostomum marginatum from the Musculature of Smallmouth Bass . . . . 53 Strigeid Metacercaria of the Black Spot in the Cyst . . . . . . . . . . . . . . . . . 6O Unidentified Strigeid Escaping from the Cyst . . . . . . . . . . . . . . . . . . . 62 Unidentified Strigeid of Black Spot Com- pletely Free from Cyst . . . . . . . . . . 64 FIGURE 12. 13. 14. Page Metacercaria of Posthodiplostomum minimum Obtained from the Green Sunfish, Lepomis cyanellus . . . . . . . . . . . . . . . . . 67 A Mature Male of Leptorhynchoides thecatus Showing the Internal Organs and Spines on the Proboscis . . . . . . . . . . . . . . . 7l Pomphorhynchus bublocolli, Entire Female Showing the Characteristic Bulbous Enlarge- ment on the Proboscis . . . . . . . . . . . 74 vi INT RODUCTI ON Animal parasites are organisms that live at the expense of other animals, usually without killing the host. Par- asites, particularly internal parasites, may decrease the health, vigor, and reproductive potential of their host. These effects are accomplished by virtue of parasitic ac- tivities inside the host tissue. Some parasites act on fish by consuming body tissues and organs, while others produce various substances which may have a toxic effect on the fish, disrupting normal metabolic processes. Still other par- asites inflict bodily, physical and biologic injuries which, in turn, give way to secondary bacterial infections. For the past several years the general public has be- come increasingly aware of fish parasites. Fishermen are interested because some parasites cause ugly sores or ul- cers in the flesh of the fish and render them unfit for fish markets. Federal and State fish and game agencies are in— terested in fish parasites because of the destruction of young fish in hatcheries and also by reason of their effects on the fecundity of game fish. Lake Manitou on North Manitou Island, Michigan, has been producing a good catch of sport fishes for the past several years. The lake still retains its position for the small- mouth bass. However, during the last few years the fishes caught have been found to be heavily infected with parasites. The parasitic survey carried out by Alexander (1959) has revealed a generalized infection of fishes with various helminth parasites including the bass tapeworm, Proteoce— phalus ambloplitis. Increasing damage was observed in small; mouth bass with increasing age. The injuries, especially in the ovaries, were so severe that it was doubtful they could have spawned. It was indicated that these fish were pre- vented from spawning by the damage caused by plerocercoid larvae of the bass tapeworm. The primary objectives, there- fore, of this investigation were to do a comparative study of the degree of damage inflicted by the larval stage of bass tapeworm in different age groups of smallmouth bass from Lake Manitou, Michigan; to determine species of hel- minth parasites present in the smallmouth bass other than the bass tapeworm; and to determine whether or not the bass tapeworm had any deleterious effect on the growth of the fish. Particular attention, however, was paid to the bass tapeworm, Proteocephalus ambloplitis, because of its high economic importance. Numerous investigations dealing with the descriptions of freshawater fish parasites have been conducted in the past. Recently, emphasis has changed from descriptive surveys to the studies of ecological and host-parasite relationship. These ecological surveys, therefore, have stressed the en- vironmental relationships between parasites and their host, work that has in many cases been complicated by the fact that many parasites require several different hosts to com- plete their life cycle. Also, for reasons unknown, some parasites show prefer- ence for a particular host—tissue over the others. The greatest difficulty encountered in these respects is that the life cycles of many of these parasites are still not completely known. It is fortunate, however, that the life cycle of bass tapeworm has already been worked out. REVIEW OF THE LITERATURE Proteocephalus ambloplitis was first described as Taenia ambloplitis by Leidy in 1887. The specimen was secured from the stomach of Ambloplites rupestgi§_(Rafinesque)from Lake George, New York. La Rue (1914) suggested that this parasite was probably the plerocercoid stage of Proteoce- Ehalus ambloplitis described earlier by Leidy in 1887. Infection of the smallmouth bass, Micropterus dolomieui Lacépede, by this species was first identified by Benedict in 1900 in Ward's collection from Lake St. Clair, Michigan. Five years later, Marshall and Gilbert (1905) mentioned it in connection with a study of the food of fishes from lakes in the vicinity of Madison, Wisconsin. In his monograph on the Proteocephalidae, La Rue (1914) examined critically all the materials available on this species. He carefully described the parasite and determined the synonomy of the forms described by Ribbenbach in 1896, Linton in 1897, and other workers. Cooper (1915), while making a systematic study of fresh- water fishes of the Georgian Bay region, noted some plero- cercoid larvae in the smallmouth bass (Micropterus a dolomieui). By comparison of the adult characters with those of the larvae, he showed that the latter was Proteo- cephalus ambloplitis. Recently, a new species, Proteoce- phalus microcephalus has been described from the intestine of Micropterus dolomieui from the northern California waters (Haderlie, 1953). The effects of helminth infections on fish has been studied by several investigators. Davis (1953) described many helminth infections and the physical damage they do to their fish hosts. Rich (1924) was one of the first to re- port the heavy infection of gonads with the larval stage (plerocercoid) resulting in complete sterilization of the adult smallmouth bass. Hunter and Hunter (1938) showed a statistically signif- icant loss in weight of smallmouth bass heavily infected with the bass tapeworm larvae. This loss of weight was partially attributed to the disturbance of metabolic proc- esses caused by the migrating larvae. Hubbs (1927) noted retardation of early growth and retention of the larval stages of fish apparently caused by heavy helminth infec- tion. Moor (1926) described in some detail the invasion of the reproductive organs of fish by the larvae of bass tapeworm, Proteocephalus ambloplitis, and indicated that fish population could be eliminated by ensuing lack of reproduction. Hunter (1942) stated that it is not known exactly how the plerocercoid larvae of the bass tapeworm causes sterility; by mechanical damage, chemical change or by a combination of both. He also stated that heavily in- fected fish swim more slowly and are easier prey for fish- eating birds. Sillman (1957) noted that fish having many metacercaria encysted in the liver suffered no inconvenience. Hunter (1937) demonstrated that if sufficient liver tissue is destroyed by the metacercaria of Posthodiplostomum minimum, the host will die. Fischthal (1953) from his experiences in the examination of northwest Wisconsin fishes reported that the condition of most fishes was not handicapped by their parasitic burden. He believed that in the dynamics of an aquatic environment there are several factors (physical, chemical, biological) which can influence the condition of a fish or fish population. Unless rigid controls are maintained, it is extremely hazardous, even after statistical analysis of the data, to claim with certainty that parasitism is the cause of poor condition. Hunter and Hunninen (1934) reported a marked difference in the position and numbers of the plerocercoid larvae of the bass tapewrom within the viscera of smallmouth bass. Their study of larval length in 90 bass of different age groups showed the mean length of plerocercoid larvae from the gonads to be always larger than the mean length of lar- vae from the rest of the organs. Many surveys of fish parasites have been conducted by state and federal agencies in recent years in the United States because of the growing emphasis on fish management. One of the earliest and most complete surveys for fish parasites was conducted at Oneida Lake, New York by Van Cleave and Mueller (1934). Many similar investigations in different areas of the United States and Canada have been conducted by Bangham (1925, 1941, and 1951). More detailed work, dealing exclusively with the bass tapeworm, Proteo- cephalus ambloplitis, was conducted by Morrison (1957) in southern New Hampshire. In Connecticut, Hunter (1942) found that fishes in three lakes were heavily infected with the bass tapeworm. He showed that the average number of worms per fish in smallmouth bass was 29 and slightly higher, 36.1, for large- mouth bass. Although the latter were more heavily infected with the bass tapeworm, only 74 per cent of them were para- sitized as compared with 95 per cent of the former group. Fischthal (1953) carried a thorough survey of fish parasites in the northwest Wisconsin waters. Over a period of three years a total of 4,532 fishes, representing 61 dif- ferent species and subspecies and collected from 124 dif- ferent lakes and streams, were examined for parasites and 4,186 or 92.4 per cent of them were found to be infected with at least one species. This survey work included 27 smallmouth bass obtained from nine different waters in which all (100 per cent) were infected and a total of 28 different species of helminth parasites were recovered from them. Sinderman (1953) found the concentration index for four heavily parasitized pond fishes in his survey area to be 53.7 worms per largemouth bass and 74.2 for smallmouth bass. Out of 20 smallmouth bass examined 95 per cent were found to be infected. This represented the highest percentage of infection of all species of fish examined by him. In New Hampshire, Morrison (1957) found the smallmouth bass index to be 80.2 worms per fish, indicating a heavy infection. However, largemouth bass from the same body of water yielded very light infections. He examined a total of 192 smallmouth bass in which 80.2 per cent were infected. Bangham (1925 and 1927), Hunter (1928), and Hunter and Hunter (1929 and 1930) have investigated the life cycle of Proteocephalus ambloplitis. Definitive hosts of this parasite thus far reported are the smallmouth bass (Micropterus dolomieui), largemouth bass (Micropterus salmoides), rock bass (Ambloplites rupestris), bowfin (Amin calva), burbot (Lota lota), and yellow perch (Perca flavescens). The adult worms are located in the upper intestine of these definitive hosts. Gravid proglottids with the char- acteristic dumb-bellshaped eggs in tremendous numbers are passed out of the host in faeces. These eggs are not very viable and must be eaten by the first intermediate host within 24 to 36 hours (Hunter, 1928). The first interme- diate host may be any of the following species of Crustacea: Hyalella knickerbockeri, Cyclops prasinus, Cyclops albidus, Cyclops leuckarti, Cyclops vulgaris and Cyclops serulatus (Hunter, 1928). The onchosphere is liberated from its enclosing mem- brane, penetrates the intestinal wall of the copepod and develops in the body cavity of the crustacean as the first larval stage (procercoid). When an infected copepod is 10 eaten by a suitable second intermediate host, which con- sists of 16 different species of fish including the small- mouth bass (Fischthal, 1953), the procercoid penetrates the stomach and intestinal wall of the fish and migrates to liver, spleen, kidneys, wall of the gut and gonads. In course of time, the larva looses its embryonic hooks, de- velops the primordial suckers with the fifth apical sucker, and is changed to the second larval stage (plerocercoid). For the larva to become an adult, the second interme- diate host must be eaten by a susceptible definitive host. In a fashion similar to that of the procercoid, the plerocercoid is digested out of the tissue of the interme- diate host. It attaches itself to the intestinal wall of the final host with the suckers on its scolex and grows into an adult tapeworm. Smallmouth bass, by virtue of their feeding habits, are exposed to mature and immature plerocercoids almost all through their life. As young fry and fingerlings, they ingest copepod members of zooplankton, some of which con- tain proceroid, and thus inadvertiently become the second intermediate host. When the fish attains a greater size, they start feeding on fry and fingerlings and may acquire the fresh infection of immature plerocercoids. 11 The life cycles of other parasites are equally as com- plicated as that of the bass tapeworm. In some cases the fish acts as an intermediate host, while in others as the definitive host. MATERIALS AND METHODS Obtaining fish sample: A total of 42 fishes were examined for different para- sites under this investigation. All these fishes were ob- tained from Lake Manitou, on north Manitou Island, Michigan. The fish were caught by hook and line during spring, 1963, quick frozen, and despatched immediately to the Michigan State University, East Lansing, Michigan. Five yellow perch, Perca flavescens (Mitchill), and one green sunfish, Lepomis cyanellus Rafinesque, were also obtained in the sample. Handling 9§_fi§h5 In the laboratory, fish were kept whole in deep freeze. They were thawed only at the time of examination. Fish were thawed individually as needed and their weight and lengths were recorded. A scale sample, from the region of the left pectoral fin, was also taken at this time. These were placed in a scale sample envelope bearing the same record number as the fish. After these parameters were recorded, the fish was ex- amined for evidence of external infection, or parasites, close to the surface of the skin. A mid-ventral incision was then made, fish eviscerated, and the sex and degree of 12 l3 maturity were noted. The head and body, including general musculature were then examined, and the parasites found placed in 0.7 per cent sodium bicarbonate solution. Viscera and gills were then separated in the following pieces: gills, stomach including pyloric caeca, small and large intestine, spleen, liver, kidneys, gonads, and the heart including big blood vessels. Each was opened up with fine scissors and placed along with its contents in petri dishes and covered with saline solution. In searching the fish for the plerocercoid larvae of the bass tapeworm, Proteocephalus ambloplitis, each part of the viscera was exposed with the use of a fine scissors and a blunt probe. These parasites, which were all dead because of freezing, were counted separately and measured under a binocular dissecting microscope to the nearest tenth of a millimeter. More emphasis was placed on the determination of the presence of these larvae in the vis- ceral organs. Smaller worms were handled with capillary pipettes, the larger with needles, forceps or wooden ap- plicators. Helminths were listed by their host number and by their location inside the host. Stomach content of the fish,vflmme found, were also examined and recorded. 14 Handling 9f_Parasites: Sodium bicarbonate solution (0.7 percent) was used for cleaning the worms as it simulates conditions in the intes- tine and tissues of the host and dissolves the mucus more effectively than the solutions of sodium chloride (Van Cleave and Mueller, 1934). Worms were washed in additional saline solution before being fixed. Larval forms of the bass tapeworm were fixed directly in a mixture of: com- merical formalin five parts, 95 per cent ethyl alcohol 25 parts, glacial acetic acid one part, and distilled water 20 parts (FAA). The larger trematodes and tapeworms were placed between two glass slides, taking care the pressure was just sufficient to keep the worms straight, but not to squeeze them. The fixative was pipetted between the two pieces of glass slides. Worms were allowed to remain in this position for 24 hours when they were finally trans- ferred to 70 per cent ethyl alcohol with 3 - 5 per cent of glycerine. Acanthocephala, that were collected for identification, were placed in distilled water for the first 24 hours and kept under refrigeration. This caused the worms to swell up and the proboscis to be extruded. They were later fixed in FAA and preserved in 70 per cent alcohol. 15 Collecting and processing of parasites causing black I spot required a somewhat more elaborate procedure. Both, ; the tissue digestion method as described by Hoffman (1955), as well as the fine dissection under a binocular dissecting microscope, were tried. Good results, however, were obtained by the later method after a little practice. Tissue digestion method of freeing encysted nematode and trematode larvae has been in use by many workers and is particularly useful in Trichinella spiralis. The di— gestion solution, as used in this study consisted of 0.5 per cent pepsin in 0.25 per cent of hydrochloric acid made up in 0.65 per cent saline solution. Digestion consisted ' of teasing the tissue with fine needles in a petri dish and then digesting for 10-15 minutes. The parasites were allowed to settle and the solution decanted followed by three or four saline washings. This procedure released the strigeid cyst by digesting away the tissue of the host, leaving behind the metacercaria in their cyst. Determination gf_the age 9; fish: Scale samples obtained from individual fish were used to determine their age according to the methods described by Lagler (1956). Processing of the scales for counting the 16 annuli consisted of pressing five or six scales between transparent cellulose acetate plates with a roller scale press. The impressions were examined under a microprojec- tion apparatus and the scale annuli were counted and re— corded as the approximate age of the fish. Identification gf_helminths: For positive identification, samples of parasites were stained and mounted on glass slides. All parasites were stained in para carmine, dehydrated, and permanent mounts were made using standard technics (Guyer, 1953). This stain yielded very good results. The mounted specimens were ex- amined under compound microscope for final identification. The main source for cestode identification was by comparing the morphological characters described by Wardle and McLeod (1952), and Yamaguti (1959); while trematode identifications were centered around the work of Yamaguti (1958), and Van Cleave and Mueller (1934). For the identification and classification of Acanthocephala, works of Van Cleave (1919) and Van Cleave and Mueller (1934) were freely consulted. During the course of investigation, several tissues of fish were also fixed and preserved in FAA solution for 17 histopathological examination. These were later passed through usual histological techniques and sectioned at 8 microns. Sections were stained with Harris' hematoxylin and eosin stain. Biometrics used: Correlation coefficients and regression coefficients were used in this study to find the degree of correlation between the rate of growth of the parasite and the host. Data were further analysed to compare the rate of growth of the parasite within the visceral organs of the small- mouth bass. Analyses of data were carried out by standard statistical procedures (Snedecor, 1959), which express the average change in the dependent variable for each unit increase in the independent variable. To test the statis- tical significance of the regression coefficients the analysis of variance method was applied. RESULTS AND DISCUSSIONS A total of 42 fishes, representing three different species were examined. These included smallmouth bass (Micropterus dolomieui), yellow perch (Perca flavescens), and green sunfish (Lepomis cyanellus). Of these 42 fishes examined, all were found to be infected with at least one 'species of helminth parasite. Altogether a total of nine different species of parasites were recovered representing the three phyla of the helminth group. Tables 1, 2, and 3 give the host-parasite index in their phylogenic sequence recovered from the three species of fishes of the sample examined. As the fishes were very heavily infected, keeping records of the absolute parastie count of the individual species was rather difficult. This was especially so with the strigeid worms causing black spot, plerocercoid larvae of the bass tapeworm, Proteocephalus ambloplitis, metacer- caria of the yellow grub, Clinostomum marginatum, and the white grub of the liver, Posthodiplostomum minimum. Fur+ thermore, in View of their generalized, heavy infection it was found unnecessary to do so. However, to determine the intensity, or degree of infection of fish with different species of helminth parasites, three categories were used 18 19 o o m.ma H.0m .H.©m o.m m.m o O o.mN m.mm H.©m mumum coauowm unmflq >>mmm use 02 -602 Nmmmn SDDOEHHmEm UUDUUMCH mo mmmucmonmm .mpwmmomq HowHEoHoo msumumonowfi .mmmm SDDOEHHmEm Eonm omnm>oowm mopflmmnmm Ahmma .wvflmqv mmHuHHQOHQEm .m Lemma .seamav mauaamoHQEm am mmma .pamacflmz msamnmmoomvonm mscmw mmma .mHoE macaamsmwuomgonm maflEmmnsm Hama .msm ma mmpflamzmmoomuonm maflfimm mNmH .maoz mmpflamnmmoomuonm nmpno mpoummu mmmHU Anomm xomanv cammauum emanaucmaac: Ahmad .mmsmsm ..mm.mmmmmwm "V «M omma .mHOQSQ ESEoumoHQHUonumom momma mama .Hfiamofluaoz mmcaeoumoamflo saflamwnsm mmma .HmHHHom mMUHEODmonHQ SHHEmm Amama .Hndaoesmv asumcflmume am omma .meflma guacamocaao mscmo .pumnm mmsHEODmOQHHU >HHEmeSm Hood .mssq mmpfleoumocflau haflemm mood .HUGSUO mumEODmomoum HmUHonsw mmma .cmpwsmm cw> mwcmmflm Hmouo MUODmEmHB mmmao mummcucHeHmnsumHm adamam Noma muflmmumm HammHSUHS .souflamz mxmq Eoum cwxma .H wanme 2O EHOB USU mo momnw Hm>umqm and mo unsoo muammnmm m .usmflq owloa mo assoc mpflmmnmm m .mpmnmpoz whoa no on mo assoc wuflmmumm m .m>mmmm pmcflfimxm mhm3 mmmn SDDOEHHwEm om mo Hmuou 4 H m.sm m.m o o mama .zpmmmz um sums muosmoHsuoo .o mama .npmmmz um UHMB mmUHODHcomo mscmw mmma .msoummammz um Mnow wmsflpflouflcomn >HH88szm vmma m.mm b.0H O O mama .UHOQQOU mmpflcmaasusu haflemm Homa .mcflmmfln mmoflnsnflmm pruo mpoumEmz mmmao mmsunfleHwnmemz asawnm .mcaxaaa Haaooonasn «m moma .fiaawoflucoz m550c>nuonafiom momma o 6.0m m.mm b.6H Aamma .aoucflqv mspmoman um thna .mmmONv mmpflosocNSHOmeq mscmw mama .ccmEmm mmpflnocmnuocflnom maflEmm mamnmwooagcmommamm HwUHO wamnmeOSDCMUMDUZ mmmau wamsmwoonpcmom Edawnm coflpomm unmaq wpmnm >>mmm -aa oz -602 Nmmmn QDSOEHHmEm omuommcfl mo mmMDGmUHmm muamwnmm Aomscaucoo H manmev 21 Lemma .moamqv mmauflaaoHnEm.4m mmma .Ucmacwmz msHmamvoomuonm mscmw mmma .mHoz mmcwamnmmuomuoum >HHEmeDm HHmH .msm MA mmpwamsmmoompoum >HHEmm mmma .mHoz mmpwamzmmoomuoum nmUHO mpoummo mmmao Anomm xomanv gammaupm BmHMHucmcha Ahmma .mmsmsm ..mm.mmmmmmm "V «M omma .mflonsm EseoumoHQHUOSDmom mocmw mama .HHHonpcoz wmcfieoumonHQ >HHEmeSm omma .umflumom meHEoumOHQHQ haflsmm Amama .Hamaoasmv asumcamnme am omma .meama aseoumoaaao macaw mood .uumum mmcflfioumocflao SHHEmwnsm Hood .mssq mmpfleoumozflau mafismm mood .umcnpo wumEoumOmonm uwpnonsm mmma .cmpmcwm cm> mmcmmfla umpno mpoumEmuB mmmav mmzucfieHmzsumam Esamam coHuomm unmaa mumum >>mmm Isa oz :60: mnonmm BOHHmm pmuommsfl mo mmmucmoumm mmuflmmnmm acmmflsoflz .souficmz mxmq Eoum amxme .AHHASUDHSV mcmomm>mam momma .Qoumm 3oaamw Eoum pmum>oomm mmuflmmumm .N manma 22 EH03 map mo mmmum am>umam mla mo assoc muammnmm m .uzmaa mvloa mo ucsou muammumm m .wumnmpoz whoa no om mo ucsoo wuammumm m .>>mmmm penafimxm man nunmm 30aamm m mo aMpou ma 0% oo o 0 0m ON 0 O hama .Summmz um puma MH0£Q0a>uoo .Q mama .aammmz um puma mwoouacomm mosmw omma .mcoummammz um xuow macaeaouchmo maaEMUnsm soma .eaonnoo mmeacmaasoso maaEME aoma .mCammaQ mmpansuamm pruo mpoumfimz mwmao mogucHEHmnumEmz asamam Aamma .coucaav.mmmmmmmm [a Amnha .meONV mUUHOSUcSSHODQUa mocww mama .csmemm mmoasos>£HOCa£om maaamm wamnmwoozucmommmamm Hmpuo mawnmooozpsmomuoz mmMaU mawnm000£ucm0< Edamnm coauoww unmaa mumnm m>mmm Isa oz loo: mnonmm 3oaamm pmuuwmca mo mmmucmoumm mmuammnmm Inmscaucou m manmev 23 m>onm cam om mo ucsoo muammnmm m EuoB may mo mmmum am>nmam um>aa Eoumv usmEDmODCa Eonmm .coauommca >>mma “meuoa mo ussoo mpammnmm m .cOauommsa mumnmpoe “mla mo unsoo muammumm m .cOauommca “smaam mamEMm may Ca Umpcwmmummu mmB Smamcsm cmmum mco maco a amma 4A a mxamma .meamqo mauaaQOHQEm .m mmma .©CMaCam3 .moamnamoowponm mssmo mmma .Maoz mmsaamsmmoomponm waaEmmnsm aama .msm ma mmpaamnmwoomponm maaEmm mmma .Maoz mwoaamnmmUOUDOHm Hmpuo mpoummo mmMaU CESHHMUUMSV EDEHCHE ESEO#WOHQHUO£#@OQ Anomm xomanv eammauum swamauamaacp Anmma .mmnmsm ..mm mmwmmmm nv mm m mmma .maonsn ESEODmOaQaUOSDmom mscmw mama .aaamUHUaoz mmaaeoumoamaa saa3mmnsm omma .umauaom mmpanumOamaQ maafimm Amama m .anmaoosmv EdamchHmE 2w omwa .wpawa ESEODmocaaU mscww moma .uumum mmcaeoumocaao saaammnsm aoma .mnsa mmpaeoumOCaaU maaEmm moma .Hmcnpo mumEoumowonm Hmpuonsm mmma .cwpmcmm sm> mwcmmam Hmono mpoumEmHB mmmau mmaucaaamnmumam Esasnm unmaa mumnwpoz >>mmm NCOauommcH cmxma .msmeCammm mmaamcmww mafiomma .smamssm cmmuw Bonn Ume>Ome mmuammumm MOHHmMHMQ acmmazoaz .soyacmz wxwa Eoum .m manma 24 which indicate whether the fish was lightly, moderately, or heavily infected. A light infection was represented by 1-9, a moderate infection by 10-49, and a heavy one by 50 and more parasites of a given species. No further counts were made beyond fifty with the exceptions of the spiny- headed and the roundworms whose numbers were comparatively low. For this reason, hunter's (1942) method for determining the parasite concentration index is not applicable in this study for all the species of parasites recovered. The fish parasites of Lake Manitou, Michigan, and their relations with the host will be discussed separately. Order: Proteocephalidea Mola, 1928 Family: Proteocephalidae La Rue, 1911 Subfamily: Proteocephalinae Mola, 1929 Genus Proteocephalus Weinland, 1858 Proteocephalus ambloplitis (Leidy, 1887) Hosts: Micrgpteru§_dolomieui, Perea flavescens, and Lepomis gyamellus A total of 36 smallmouth bass were examined. Of these 36 fish examined, all were found to harbor at least two kinds of worms--the plerocercoid larvae of the bass tape- worm, Proteocephalus ambloplitis, and the mature stages of the spiny-headed worm, Leptorhyncoides thecatus. The sexually mature stage of the bass tapeworm, 25 Proteocephalus ambloplitis, was recovered from the pyloric caeca and the small intestine of smallmouth bass. As many as 50 adult worms were recovered from five of the bass examined. Adults of the genus are recognized by the fol- lowing characters: Scolex unarmed, with four large, round or oval typical suckers directed outward and anteriad (Figure l). A fifth or apical organ may or may not be present. Excretary stems are situated slightly medial to the outer edge of medulla. Testes, 70-100 in number, are situated between vitellaria in one contineous layer dorsal to uterus. Ovary is bilobed, extending transversally at posterior end of proglottids. Vitellaria are in lateral fields of medulla outside of excretary stems. Segments of the proglottids are jointed to each other with rounded ends (Figure 2). The life cycle of the bass tapeworm is discussed earlier in this paper (page 9). Identification of the plerocercoid larva is based on the presence of the fifth sucker. Even after the scolex is evaginated, the vestigial fifth sucker is in evidence where it may be seen embedded in the scolex near its distal extremity (Figure 3). The plerocercoid larvae recovered from the lumen of the gut retained the 26 Figure 1. Unarmed scolex of Proteocephalu§_ ambloplitis (adult). 125 x, 28 Figure 2. Mature proglottid of bass tapeworm, Proteocephalus ambloplitis showing the position of uterus, testes, and Vitelline glands. 135 X. 30 Figure 3. Larva of Proteocephalus ambloplitis (Plerocercoid) with characteristic vestigial fifth sucker embedded distally in the evaginated scolex. 6.5 X. W. k. — .‘W rm. Rm}... II.‘ 32 everted scolex (Figure 4). The fifth sucker of the plerocercoid larvae may even persist for a time in the adult worms. The 36 smallmouth bass examined in this study ranged from 7.3 to 19.5 inches in length. Smallmouth bass meas- uring from 10-12 inches were most heavily infected with the plerocercoid larvae, whereas the younger specimens yielded fewer numbers of parasites. As expected, the larger bass heavily infected with the plerocercoid larvae contained an abnormal quantity of connective tissue in their coelom, which made it difficult in most of the cases to recognize or to separate the visceral organs. Most of the larvae were found encysted in the mesenteries or embedded in the heavy network of proliferated connective tissue. This proliferation of connective tissue was apparently associated with the damage caused by the internal migration of these parasites. Also, in such locations, a heavy fibrous cyst was found thrown closely around the developing plerocercoids. Larvae located in the liver, spleen, and gonads, however, were found loosely encased in thin, membranous cysts (Table 4). Hunter and Hunninen (1934), based on their previous experimental findings, indicated that the mesenteries and omenta constitute, beyond doubt, the first location 33 Figure 4. Larval stage of bass tapeworm, Proteocephalus ambloplitis (plerocercoid) with everted scolex. The fifth vestigial sucker is situated in the center. 6.5 X. 35 Lemma .amsaaamv coauomm Isa 0H0>0m m0 mommo mammuo may no mommnsm esp so pmumooa maumOE mummo .ummu mzu 00am Ina Umpaom muammuwm Ga Smam wnu aaax no one HOaHmumom mnu cmwamm .Hmawmwa mmE .mmsmmau Hmnuo £ua3 ummo DcmummmCMHu 0cm .mwaumu Amamcsm ESEoumOam 0cm um>aa mwouummn wuas3 UmaamBIcaEB IcmmmE .Hm>aa cmmuo Iammmwmmm coauosuumno amoaCMLU IwE an GOaDQHOQO mcmunEmE mSOUSE 8cm coaummmao rua3 any on omromuum mummumuca mmE .coauomm mammOOa xwaoom nua3 Isa >>mmn :H .umos pmmeE aa03 mucmE Ca musmanusc Damp Immm .Umoun .E0 «.0 InomEa m0 coaumammp 0D a.o paw mcoa .Eo mcapmwuca aamEm mauaamoaQEm wmsmo mmE .Q3ocx Na on ma mcanommu map m0 coaunom mmmn msamnmwu Doc mmoa0£umm Domxm EH03 mua£3 Cane Momma paw 00000 ausoEaamEm Immmmmm moauuoamoum 0: .xmaoom 05¢ m0 xwmm mag :0 meosm mmummu SmaMGSm amamaum0> Spmam m can one mwaum>0 cmwum 0cm Amgma .Hmucsmv mnmxosm w an Umuaumu .aam3 Dam .noumm 30a mawaaQOaQEm mmmn zusoEaamEm lumumso .mcoa .EU 0 .Hm>aa .zmmamm lam» .mmmn a mmflmmmmw ca wuaaaumpm momsmo ou m5 EH03 0Da£3 Gaza .mmanmpsmmmE CH SpsoEaamEm lawmmmm mosmuuomEH mmusummm m>apman0mwa nmamafi scaumooa umon amam muammumm umom swam may on coaumamm sua3 mmuammumm cmmanoaz nucaanm m0 mumEEsm 0>aumanomwn .souacmz wxma Eonm meme .w magma 36 muamMHmm 03H m0 wmmum am>Hma* Hmmma msousEnsm paw msoosE may OHGa mammmp UmppmnEm mau ImOQOHm mzp m0 Qasn mmmmmmma Eua3 Ham3 Hmaaummpaa mmpaoonwzHoumma msu 0H vwsomu aaaOUOQaSQ How UmnaHommo IHm wamEOHum EH03 mcaummpsa aamEm mmmn mmmmmxmm Hmnu 0H HmaaEam EHomaHmm 0Ha£3 Gaze m0 coauHom Hmmmb QHSOEaamEm Immmamm aam3 Hum m0 Hmmma msoosE coaHmHoQO boom £Ha3 may 0H ownomuum Unapmmuca 0H0MHcha mmE .aam3 maEHam .mCOa .EU aamEm m0 coauHom mmmmwwmm Ham mnu m0 meazaa m.o 0H m: mcasommH HoaHmHsm Ucm mmmn mmpaosocwsH msoosE map mHsncH .EH03 0Ha£3 paw canfi .mowmo .zumEoum SHDOEaamEm Immmmm mocmuHomEH mesumwm m>aumaHommn Amamafi.coaumooa Hm0£ Swab mnammHmm Howscaucoo a manmev 37 normally assumed by the young, migrating larvae of the bass tapeworm. With the growing age of the bass, the larvae tend to migrate and occupy the more favorable and better developed sites like ovary, testes, liver and spleen. This observa- tion was further exemplified when the younger bass of the Lake Manitou, Michigan, were found to harbor either fewer numbers or no parasites in their spleen and gonads, although their mesenteries and omenta were beaded with several small larval cysts. It is presumed, therefore, that the initial invasion of these larval forms elicit a severe host re- action resulting in a heavy fibrotic cyst in the first instance as compared with the later thin, membranous cysts in the liver, spleen, and gonada mentioned earlier. As can be seen from the forthcoming, Proteocephalus ambloplitis affects the various species differently. This is true not only for the type of infection but also the degree of infection. The plerocercoid larvae in small- mouth bass and green sunfish were generally located near the surface of the organ. In heavily infected fish they were found throughout the visceral tissues. Extensive de- generation of the liver tissue caused by the migrating larvae of the bass tapeworm may be seen in Figure 5. Similar degenerative changes were also noticed in the 38 Figure 5. Section of liver of smallmouth bass showing extensive degeneration of the Liver Tissue. Numbers 1 and 2, on the photograph, show the cross section of the parasite. Hematoxylin-eosin. 370 X. 4O ovaries of the smallmouth bass permeated with large numbers of the plerocercoid larvae. Eggs in this organ were de- generating and the whole organ was wrapped around a thick fibrotic mass of connective tissue. Apparently, the fish were rendered sterile by the combined effect of the me- chanical injury caused by the parasite and the response of the host fish to this infestation. Comparison gf_the plergcercoid larvae ig_y9ung and adult smallmouth bass: The sample of 36 smallmouth bass examined ranged from two to ten years in age and was composed of 20 males and 16 females. The 20 females ranged from 2 to 10 years, while the 16 males represented the age group of 3 to 9 years. Table 5 shows the distribution of the calculated mean length and the standard error of the plerocercoid larvae in the visceral organs of the smallmouth bass. Figures in parenthesis represent the total number of parasites examined in each case. There were 414 plerocercoid larvae measured from ovaries and testes and 885 from the rest of the visceral organs. The overall average length of plerocercoids in the visceral organs of the smallmouth bass, regardless of the age group of the fish, are shown in Table 6. The l 4 vmsamem mmHHmMHmm m0 HmnEsc amuou may ucmmmHmmH mmmwaucmHmm Ca mmHsmam ..m.m + saw: u.» A ma V A va V A m V A ma V mm. H fim.m wm. H m¢.o «v. H mo.¢ mm.m H ma.ha a x A AA V A mm V A «a V A Ha V A om V on. H ha.© ha. H m¢.m mm.a H ao.© mm.a H am.® ¢¢.a H m¢.ma N xH A ma V A m V A m V A on V am. H aa.m om.a H 08.0 oo.a H ¢m.¢ boa. H oo.ma a HHH> A aa V A m V A m V A om V am. H hh.a ma. H mm.m mm. H om.~ ma. H m@.@ a HH> A ma V A m V A Ha V A mm V mm. H mm.N ¢©.a H 00.0 nfi. H mm.v on. H mo.m a H> A an V A s¢ V A oa V A om V mm. H m¢.m ma. H mm.a ma.a H mo.¢ mm. H hm.m v > A om V A mma V A mm V A we V A we V am. H nm.m om. H mm.a mm. H mm.m ma. H hm.m mm. H ao.n m >H A ma V A msa V A 64 V A moa V A moa V ¢m. H am.m 50. H mm.a mm. H N¢.m ma. H mm.m Na.a H vm.m va HHH A Hm V A Ha V A ma V A ma V ma. H om.a mm. H «N.N mm. H vm.m Hon. H mo.v m HH mwumwe >H0HG0m02 cmmamm Hw>aa mHm>O owcamem ODOHm A.EEV ncmmHo chmemap Ga mm>HMa m0 flnmcwa Smam m0 HwnEsz 00¢ mnu ca mauaaaoanEm.mmamsamoomMOHm m0 mw>qu paoonoonam m0 npmcwq meHm>¢ mQSOHO 0m< mDOHHm> m0 mmmm HHDOEaamEm mo mammHo aMHmoma> .m wanna 42 Table 6. Comparison of the Plerocercoid Larvae of Proteo- cephalus ambloplitis in the Visceral Organs of the Small- mouth Bass Taken from Lake Manitou, Michigan, Irrespective of Their Age Group Total number Average length Organs of parasites of parasite examined (mm.) A — Ovary 307 8.44 i .36 B - Liver 250 3.53 f .15 C - Spleen 130 4.17 i .25 D - Mesentery 505 1.83 f .04 E — Testes 107 3.62 i .18 * Calculated probability of comparisons between ovary and other visceral organs: A vs. B: P < 0.001 A vs. D: P < 0.001 A vs. C: P < 0.001 A vs. E: P < 0.001 43 contrast is striking and the comparison of the average length of plerocercoids in ovary is statistically signif- icantly greater than the average length of plerocercoids in the rest of the organs. The consistent increase in the mean length of plero- cercoids in the ovary and the testes with the gradual in- crease in the age of the fish is again striking. A correlation of .96 was obtained between the average length of plerocercoids in the ovary and the increase in the age of the fish by one year. This is statistically highly significant. Similarly, correlation between the mean length of larva in the testes and the increase in the age of the fish by one year is .94 which again is highly signif- icant statistically (Table 7). In order to compare the growth rate of plerocercoids in the ovary with those growing in the testes, separate regression coefficients were obtained for each of these organs. The regression coefficients of lengths of plero- cercoids in ovaries and testes are 1.60 and 0.55 respec- tively.‘ By F test, each of these regression coefficients were found to be significantly different from that of a simple chance occurence. The average length of plerocercoid 44 Table 7. Correlation and Regression Coefficients Between the Lengths of Plerocercoid Larvae of the Bass Tapeworm Growing in Ovary and Testes and the Annual Increment in the Age of the Smallmouth Bass Sum of squares and products Organ D.F. r b S x2 S xy S y2 l 1 Ovary 6 58.87 94.10 163.71 .958 1.60 2 2 Testes 2 20.75 11.51 6.58 .941 .55 1Significant at the 1 per cent level 2Significant at the 5 per cent level 45 larvae for a given age of fish can be calculated from this regression coefficient. Figures 6 and 7 show the equation of regression coefficient (Y = a + bX) and the calculated lengths of plerocercoid larvae in ovaries and testes. The vertical lines represent the distribution of standard er- rors for each of the larval lengths calculated. From the above statistical analysis of the data it is evident that as the fish increases in age the gonad larvae also increase. The rate of growth of larvae, however, differ in male and female gonads. The average annual in- crement in the length of larvae of testes is .55 mm. as compared to 1.60 mm. of the larvae growing in the ovary. This increase is approximately three times greater than the other. Since the growth of the larvae in the gonads is directly associated with the degree of damage done to these organs, it is apparent that extensive damage would be expected earlier in the female than in the male bass. Hunter and Hunninen (1934) did a similar type of work in smallmouth bass obtained from rivers and lakes of New York. Their study on 116 smallmouth bass revealed the number of plerocercoid larvae to be always greater in gonads than in any of the visceral organs. Also, they found the mean length of plerocercoids in gonads as 10.7 mm. and 46 Figure 6. The relationship between the age of the fish and the length of plerocercoid larvae of Proteo- cephalus ambloplitis growing in the ovaries of small- mouth bass. Vertical lines represent the distribution of standard error. Length of larvae (mm.) 20» 47 .22 + 1.60 X .96 Age of the fish (years) 48 Figure 7. The relationship between the age of the fish and the length of plerocercoid larvae of Proteo— cephalus ambloplitis growing in the testes of small- mouth bass. Vertical lines represent the distribution of standard error. Length of larvae (mm.) 49 .4)- Age of the fish ~ + -w..-. ‘vhr— .. A c—‘w.“-“~ _.—‘4F‘u—l 6 9 (years) 50 from the rest of the visceral organs as 6.2 mm. They, how- ever, did not find any correlation between the age of the fish and the growth of larvae in male gonads. One of the original proposals, besides the one dis- cussed thus far, in this investigation was to know whether or not infection of bass tapeworm had any effect on the growth of the smallmouth bass of Lake Manitou, Michigan. This consideration had to be abandoned when every fish of the sample was found to be infected with the plerocercoid larvae of the bass tapeworm. There are several variables in the dynamics of aquatic environment which affect the condition of fish (Fischthal, 1953). Under the circum- stances, comparisons, as evidenced by lengths and weights of a non-parasitized fish from different body of water will not be directly comparable. It is surprising, however, to note that, even under such heavy infestations, the growth rates of the smallmouth bass from Lake Manitou, Michigan, compared favorably with growth rates of smallmouth bass taken during a survey of 400 Michigan lakes (Beckman, 1946). Damage in these fish, based on gross and histopathological examinations, seems to be very extensive to the vital organs. However for the anglers, these fish do not show any apparent effects of 51 parasitism. They faught well on hook and line. Every fish of the sample examined had good deposit of peritoneal fat. Yellow perch of Lake Manitou, Michigan were not found as one of the final hosts of the bass tapeworm, Proteo- cephalus ambloplitis. The larval stage of the worm, how- ever, were recovered from all five yellow perch examined. As indicated earlier, infection of plerocercoid larvae of Proteocephalus ambloplitis presented altquther a different picture in this species of fish. The larvae,unlike those found in smallmouth bass, were enclosed in round, yellowish- white cyst with a heavy cyst wall. These cysts were of varying size and were distributed in liver, spleen, mes- enteries, and omenta. In no case cysts containing plero- cercoid larvae were found inside the ovary. Out of the five fish examined, only one had heavy infection. Bangham (1941) reported that the heavy cyst wall in case of yellow perch appears to retard the growth of the larva. Order: Digenea van Beneden, 1858 Suborder: Prosostomata Odhner, 1905 Family: Clinostomidae Luhe, 1901 Subfamily: Clinostominae Pratt, 1902 Clinostomum marginatum (Rudolphi, 1819) Hosts: Migropterus dolomieui, Perca flavescens, and Lepomis gyanellus. 52 Immature stage of Clinostomum marginatum (metacercaria) commonly known as yellow grub were found distributed in the general musculature, gill, gill cover, and operculum of the smallmouth bass (Figure 8). Of the 36 smallmouth bass examined, 26 or 72.3 per cent had light to moderate infec- tions. Infection with metacercarial stage of Clinostomum marginatum has practically been reported from all species of freshawater fishes except trout (Hunter, 1942). nggg_ flavescens is, by far, the most common host reported by Van Cleave and Mueller (1934). In the yellow perch this parasite had a congregation along the head, throat, gills, gill covers, and opercular regions. In the smallmouth bass and the green sunfish the cyst of Clinostomum marginatum were more common in general body musculature and not con— centrated in any particular region. However, there appears to be very little damage done to the host fish by yellow grub. According to Fischthal (1953), heavy infection of Clinostomum marginatum may increase the oxygen requirement of the fish. Life cycle of Clinostomum marginatum, along with the life cycles of other parasites recovered in this study are summarized in Table 8. 53 Figure 8. Metacercaria of Clinostomum marginatum from the musculature of smallmouth bass. 2PX. ‘2'..- .‘-- -4- -. .".‘p.. 55 mH0£H0 msoaHm> 0cm .ch309 .nonm 30aa0> .mmmn SHDOEaame cam mema .mmmn xUOH AommaV mchsaocH .Smaw cmEmmom mpHaQ mCHHmwlnmam mo mwaowmm chHmm a AnmmHV mnpuummaosoamm [HHH mm ca ammo aseflaae Hmucsm m0 wcaummucH I00 waHmonomumz wcaH>m mmmnm ESEoumoamanpumom Amcouoo asaansmV maamcm GEMa CH omam paw .mHmpcmEmamm AmmmaV mUHaQ_mcaummicmam .Amcmauam_wcmmV GOHM Hmuczm m0 mammsmomw Ucm .Lmam m0 mmaommm hm ESHmcamHmE e Hmucsm mua>mo amoosm ca Hmwucw mm>Hma mHHumsamm mmeNa ESEopmocaao Ammmmo aam ca Hmoz wumapwEkucH 03H 0H03 zmam "mnowamHBV amcam hHommmoom acoumm >H0mmmuom umHHm 00c0H0m0m umom wuamMHmm Emmanoaz .souacmz 0xma mo m0£mam 0gp EOHH Umcamuno mmuamMHmm nucaEamm m0 m0a0>0 0maa .m manma Aomma a mmmaV HOUEDE w Hmucsm .AmmmaV Hmacsm “A33 8 mmmaV Emnmcmm 56 AmmmaV ausmmEmw 8 .AhvmaV mosmsucma “AmmmHV Hchsm w Hmucsm £0H0m 30aa0m 6cm .HOQHDQ .cam I30Q .mmmn x00H .mmmn SHSOEmmHMa 0cm aamEm Aumoa amcam 03H mm aa03 mm oumavaHchH mouan mcaumwngmam mmmn £HSOEaamEm mca updaoca mmzmam m0 mmaommm Hcmemmap ma nonnaosa Hmon wHommmoum pcoomm mH0£H0 msoaHm> cam mmmfl £H50EaamEm 0cm mmHma .mmmn xUOH .anMCSm m0 mmaowmm HmOE Ca maHMUHmomumE m0 ucmEumhocm mDHMaSHHmm .mmmflme cam .mwmmmflmw mQOaUNU .HHmeosma mQOaUNM .mswanam mamaomw .mscamHmQ m00a0>0 .ameoon Imeoacx waamam>m 0:» Anon mum; swam .mmmwmm am_Ucm EDHmascmmEmo am .ma>a0>HHH mammmawm mauaaaoaQEm msamzmmuomHOHm umwoummoV A.mm mmwmmmm nV amamamm ImOaQawosumom mEH03 GammaHHm mosmHmmwm Hmaam >Homm000m pcoomm hHommmuom HmHam Hmom wvamMHmm Aumscauaoo m maanV 57 Hmon AmmmaV 00>a0>ca amcam 03H E0Hm Haaooonasn amnpnomam 0H0 mmaommm HcmHmmmaU am @0HMHmm0m H02 womanmE< mdzocmmHomEom AhwmaV 0>mmao cm> U0>a0>ca 0Hm mwaommm on w “AmwmaV .0>mmao 00> paw 0E00a0caq 0>m0ao cm> 0H mCHUHouom Aswan w mEooaocHa mo mwaowmm hm UmuHommH Hmon AAmmmaV am£u£omam Av0>a0>ca 0H0 aMCam 03H EOHH msvmomnu amsusomam mmnmam m0 mwaowmm aMH0>0m @0HMHmm0m uoz womanmE¢ mwpaonocmeouqu Awamnmwoonucmo